Citation: Moubasher H, Abd El-Ghani M, Al-Wakeel S and Bahoor A. Chemotaxonomic Significance of Flavonoids in Some Species of Galium (Rubiaceae) from Libya. Austin J Plant Biol. 2016; 2(1): 1014.

Austin J Plant Biol - Volume 2 Issue 1 - 2016ISSN: 2472-3738 | www.austinpublishinggroup.com Moubasher et al. © All rights are reserved

Austin Journal of Plant BiologyOpen Access

Abstract

The flavonoid profiles of five Libyan Galium L. (Rubiaceae) species collected from different localities and habitats were investigated. Fourteen flavonoid compounds were isolated and identified using the direct comparison of chromatographic and UV spectral analyses with standard samples. The flavone compounds were identified as apigenin (1) and its 7-glycoside (2); luteolin 7-diglycoside (3), diosmetin (4) and its 7-monoglycoside (5), as well as its 7-diglycoside (6). In addition, the detected flavonol compounds were kaempferol 3-glycoside (7), 3-diglycoside (8) and 3,7-diglycoside (9); quercetin (10) and its 3-glycoside (11), 3-rutinoside (12), 3,7-diglycoside (13), and 3-diglycoside-7-glycoside (14). The chemotaxonomic relationships of the studied species of Galium and their significance were also discussed.

Keywords: Chemosystematics; Galium; Rubiaceae; Flavonoid glycosides; biochemistry; Multivariate analysis

the Al-Jabal Al-Akhdar Mountain landscape comprises the richest vegetation and the highest number of species known from Libya [16]. The geographical affinity of the flora is mainly East Mediterranean rather than neighboring regions of North Africa [17,18].

In Libya, Rubiaceae is one of the eight species-rich families which represented by 50 genera and 90 species [19]. The genus Galium is represented by 10 species; G. mollugo L., G. spurium L., G. aparine L., G. tricornutum Dandy, G. verrucosum Huds., G. parisiense L., G. setaceum Lam., G. recurvum Req. ex DC., G. cossonianum Jafri, and G. murale (L.) All.

As from the beginning of 1990s, the increasing interest of molecular approach in taxonomic studies, especially those based on nucleic acids sequences, a remarkable decrease in number and importance of investigations dealing with chemotaxonomic evidence. Therefore, some authors urged this trend, and recommended research work on non-molecular evidences [20].

There is often confusion between different samples of this genus in herbariums and so the aim of this study is to develop a method to find characters providing data of both taxonomical and pharmaceutical use. No phytochemical studies were reported so far from Galium species in North African countries. The present work aims to establish foliar flavonoid patterns of five Galium species from Libya, in an attempt to determine chemical affinities among species and compare the results obtained with affinities indicated by evidences from morphology.

Materials and MethodsPlant material

Table 1 shows locations, types of habitats, dates of collection, GPS coordinates of localities, elevation and number of populations that were examined for the selected 5 species of Galium. Aerial parts of all plants were collected from natural populations. For each species at least two populations from distant habitats were studied. Altogether,

IntroductionGalium L. is one of the largest genera of Rubieae (Rubiaceae) with

more than 400 species included into 16 sections containing annual and perennial herb that are distributed in temperate and tropical regions of the world [1,2]. Certain species of Galium are found even in the Arctic zone or high elevations on tropical mountain ranges. Galium itself is problematic taxonomically, because taxa from different sections exhibit similar habit, many species are widely distributed and polymorphic, and species groups often are poorly differentiated both morphologically and geographically [3].

The flavonoid chemistry of Galium have been studied, and reported to contain predominantly flavones and flavonol aglycones and their glycosides. Earlier study by [4] isolated rutin and a mixture of flavonoids and diosmetin from Galium palustre L. Also, the glycosides of quercetin, luteolin, apigenin and kaempferol were isolated from Galium aparine [5,6]. Reported that Galium has long been known to contain substantial amounts of anthraquinones, with the roots being especially rich sources of these secondary metabolites. Bedstraw species, including G. mollugo, contain mollugin [7], flavonoids [8], coumarins, phenolic acids, and iridoid glucosides [9]. Recently, two new flavonoids; diosmetin glycoside and biflavone, were isolated and identified in G. verum L., in addition to isorhamnetin and its glycosides, kaempferol, quercetin, diosmetin and its glycosides [10,11].

Bedstraw species, including G. mollugo, also contain mollugin [7], flavonoids [8], coumarins, phenolic acids, and iridoid glucosides [12,13]. Some of these compounds have allelopathic, fungistatic, or repellent effects, and may also be used to flavour food or wine [14]. Recently, extracts from this plant were evaluated for their anti-cancer and anti-malarial activities and for their ability to inhibit HIV–1 reverse transcriptase, but initial results showed no activity [15].

The flora of Libya is not rich in the number of species; however,

Research Article

Chemotaxonomic Significance of Flavonoids in Some Species of Galium (Rubiaceae) from LibyaMoubasher H1*, Abd El-Ghani M1, Al-Wakeel S1 and Bahoor A2

1Department of Botany and Microbiology, Cairo University, Egypt2Faculty of Science, Al-Marqeb University, Libya

*Corresponding author: Moubasher H, Department of Botany and Microbiology, Faculty of Science, Cairo University, Giza 12613, Egypt

Received: July 22, 2016; Accepted: September 06, 2016; Published: September 12, 2016

Austin J Plant Biol 2(1): id1014 (2016) - Page - 02

Moubasher H Austin Publishing Group

Submit your Manuscript | www.austinpublishinggroup.com

80 specimens were collected from their natural habitats in different locations of Libya. Voucher specimens of the studied species were deposited at the herbarium of Cairo University (CAI).

Biochemical proceduresThe whole plant of Galium was dried in the shade and ground.

The powder was extracted with 70% MeOH three times at room temperature and evaporated under reduced pressure. The aqueous layer was stored in a refrigerator until needed to test the presence of flavonoids in different samples.

The separation of flavonoid mixture was detected by spotting all the flavonoid extracts using one-dimensional paper chromatography on Whatman No. 3mm along with the standard samples, water, 15% acetic acid (acetic acid: water; 15:85) and BAW (n-butanol: acetic acid: water; 4: 1: 5) upper phase as solvent systems. After drying the paper chromatograms, the developed spots were examined under UV light at a wave length of 365nm using an Ultraviolet Lamp (Model, UV GL-25) then in the presence of ammonia fumes.

The qualitative analysis of the crude plant extracts containing complex mixture of flavonoid compounds was carried out on Whatman No. 3mm chromatographic paper, using the solvent system BAW in the first run and 15% acetic acid in the second. The chromatograms should be dried in the hood between the first and the second chromatographic runs. All the flavonoid spots on the dried chromatograms were detected under UV light with and without the presence of ammonia.

The separation of a flavonoid mixture was achieved by elution techniques using one-dimensional paper chromatography on Whatman No. 3mm. The used solvent system in purification of flavonoid mixture was selected after preliminary one-dimensional runs in different systems to observe which has effectively separated the mixture. The used solvent systems were BAW (n-butanol: acetic acid: water; 4: 1: 5) upper phase; 15% acetic acid (acetic acid: water; 15:85) and water. The crude plant extract was applied as a band at 10cm from the top of the chromatograms and developed in the selected solvent. UV–detectable bands observed on dried chromatograms with and without exposure to ammonia fumes were cut out and

eluted with 95% methanol. Each methanolic extract was concentrated by evaporation on rotary evaporator. Additional purification of the isolated flavonoids was carried out until a pure flavonoid compound was obtained.

Ultra Violet (UV) spectral analyses were performed according to standard procedures performed by Mabry et al. [21] and Mabry and Markham [21].

The diagnostic reagents used for the UV spectral measurements of the isolated flavonoid compounds were: Sodium Methoxide (NaOMe), Aluminium Chloride (AlCl3), Hydrochloric Acid (HCl), Sodium Acetate (NaOAc), and Boric Acid (H3BO3). The absorption spectra were measured in methanolic solution against methanolic blank using automatic recording Spectrophotometer (UV–3101 PC, UV-VIS-NIR) scanning Spectrophotometer using standard Quartz cuvettes of 1cm path length. A fresh stock solution of 0.1mg pure flavonoid in about 10ml spectral methanol was prepared and adjusted on the spectrophotometer so that the major absorption peaks between 240 and 460nm. Then, the following steps were carried out:

1. The MeOH spectrum was recorded at normal scan speed, using 2-3ml of the fresh stock solution.

2. The NaOMe spectrum was recorded immediately after addition of 3 drops of NaOMe stock reagent to the solution used for step (1). After 5 minutes, the spectrum was rerun to check for any decomposition in the compound, this solution was discarded,

3. The AlCl3 spectrum was recorded immediately after the addition of 6 drops of AlCl3 to 2-3ml of the fresh stock methanol solution,

4. The AlCl3/HCl spectrum was recorded immediately after addition of 3 drops of stock HCl reagent to the cuvette containing AlCl3 used for step (3), the solution was then discarded,

5. The NaOAc spectrum of the flavonoid was determined by adding enough anhydrous NaOAc to the cuvette containing 2-3ml fresh stock solution of the flavonoid. After shaking, recording out within two minutes and then after 5-10 minutes to check for any decomposition of the compound,

Species Localities Habitats Elevation (m ASL) GPS coordinates

Galium aparine L. Gasr Libya (S4) Mountainous areas 280.7 32° 37’N, 21° 23’E

Shahat Ruins (S1) Mountainous areas 560.2 32° 50’N, 21° 55’E

Wadi Qaam (S12) Coastal wadis 20.7 32° 28’N, 14° 25’E

G. murale L. Wadi Elkouf (S5) Inland desert wadis 264.6 32° 41’N, 21° 33’E

Sharshara (S8) Canal banks 327.4 32° 27’N, 13° 37’E

G. setaceum Lam. Wadi Derna (S6) Inland desert wadis 85.3 32° 42’N, 22° 36’ E

G.tricornutum Dandy Lamluda (S2) Farmlandsbarley fields 668.1 32° 44’N, 22° 06’E

Almansoura (S3) Farmlands NA 32° 50’N, 21° 55’E

Stwa (S7) Farmlands 566.9 32° 49’N, 22° 09’E

G. verrucosum Huds. Wadi Qaam (S11) Coastal wadis 20.7 32° 28’N, 14° 25’E

Almansoura (S9) Farmlands NA 32° 50’N, 21° 55’E

Wadi Derna (S10) Inland desert wadis 85.3 32° 42’N, 22° 36’ E

Table 1: List of studied species with binomials, habitats, localities, collection dates and GPS coordinates of localities. Figures between parentheses refer to the population numbers. NA=Not Available.

Austin J Plant Biol 2(1): id1014 (2016) - Page - 03

Moubasher H Austin Publishing Group

Submit your Manuscript | www.austinpublishinggroup.com

6. The NaOAc/H3BO3 spectrum was recorded after addition of anhydrous H3BO3 to the cuvette which contained NaOAc used for step (5).

Multivariate analysis, using the species analyzed as Operational Taxonomic Units (OUT) and the flavonoids as characters, was carried out with PAST ver. 2.11 [22]. The resulting UPGMA dendrogram is shown in (Figure 1). Principal Components Analysis (PCA) was performed sing MVSP ver. 3.1 [23] and the resulting biplot is shown in (Figure 2).

Results and DiscussionAccording to recent modifications to the classification of Galium

[23], the 5 studied species are included in two sections: Sect. Kolgyda (Galium murale L., G. tricornutum Dandy, G. aparine L. and G. verrucosum Huds.) and Sect. Jubogalium (G. setaceum Lam.).

Fourteen flavonoid compounds were detected in the five Galium

species collected from different locations of Libya. Identification of the isolated flavonoids was mainly based on the direct comparison of chromatographic and UV spectral studies with standard samples. The present data clearly established that the investigated species have a simple and highly substituted flavone and flavonol compounds including flavonoid, aglycones, glycosides and methylated compounds. The isolated flavonoids based on six flavones and eight flavonols. The flavone compounds identified were apigenin (1) and its 7-glycoside (2), luteolin 7-diglycoside (3), diosmetin (4) and its 7-monoglycoside (5), as well as its 7-diglycoside (6). In addition, the detected flavonol compounds were kaempferol 3-glycoside (7), 3-diglycoside (8) and 3,7-diglycoside, (9) quercetin, (10) and its 3-glycoside (11), 3-rutinoside (12), 3,7-diglycoside (13), and 3-diglycoside-7-glycoside (14).

Identification of the isolated flavonoidsPaper chromatography: The colour reaction of the aglycone

0.32

0.4

0.48

0.56

0.64

0.72

0.8

0.88

0.96

Sim

ilarit

y

S7

S2

S9

S10

S3

S11

S5

S8

S1

S12

S4

S6

Figure 1: Dendrogram of Galium sp. based on flavonoid distributions and UPGMA clustering method. A, B and C are the three separated groups. For species abbreviations, see (Table 1).

PCA

Axis

2

PCA Axis 1

APIGENIN (ApI)

API-7-O-MONOGLYCOSIDE

LUTEOLIN-7-O-DIGLYCOSIDE

DIOSMETIN(Dio)

DIO-7-O-MONOGLYCOSIDE

DIO7-O-DIGLYCOSIDE

KAEMPFEROL-3-O-MONOGLYCOSIDE

K-3-O-DIGLYCOSIDE

K-3,7-O-DIGLYCOSIDE

QUERCETIN(Q)

Q-3-O-MONOGLYCOSIDE

Q-3-O-RUTINOSIDE(RUTIN)

Q-3,7-O-DIGLYCOSIDE

Q-3-O-DIGLYCOSIDE-7-OMONOGLYCOSIDE

-0.09

-0.18

-0.26

-0.35

-0.44

0.09

0.18

0.26

0.35

0.44

-0.09-0.18-0.26-0.35-0.44 0.09 0.18 0.26 0.35 0.44

S6

S4

S1

S12

S7

S2

S3

S9

S10

S11

S5

S8

Vector scaling: 0.87

Figure 2: PCA scatter biplot showing the relations between the separated compounds and the studied species (S1-S10). A, B and C are the separated groups in (Figure 1).

Austin J Plant Biol 2(1): id1014 (2016) - Page - 04

Moubasher H Austin Publishing Group

Submit your Manuscript | www.austinpublishinggroup.com

No. CompoundsRf value ×100 Colour reaction

BAW 15% acetic acid UV UV/NH3

1 Apigenin 92 2 P P

2 Apigenin-7-O-glycoside 47 27 P YG

3 Luteolin-7-O-diglycoside 33 29 P Y

4 Luteolin-4`-O-Me (Diosmetin) 76 4 P dP

5 Diosmetin-7-O-glycoside 34 40 P dP

6 Diosmetin-7-O-diglycoside 20 63 P dP

7 Kaempferol-3-O-glycoside 57 35 P Y

8 Kaempferol-3-O-diglycoside 35 60 P Y

9 Kaempferol-3,7-O-diglycoside 25 70 P Y

10 Quercetin 69 3 Y brit Y

11 Quercetin-3-O-glycoside 56 35 P Y

12 Quercetin-3-O-rutinoside (Rutin) 33 46 P Y

13 Quercetin-3,7-O-diglycoside 24 72 P Y

14 Quercetin-3-O-diglycoside-7-O-glycoside 16 81 P YG

Table 2: Paper chromatographic data of the isolated flavonoid compounds. Y=yellow; P=purple; dp=dull purple; YG=yellow green; britY=bright yellow.

Compound MeOH NaOMe AlCl3 AlCl3/HCl NaOAc NaOAc/H3BO3

(1) Apigenin

336 392 382 381 340 337

268 325 345 342 302 sh 269

. . . 275 301 300 269 . . .

. . . . . . 277 278 . . . . . .

(2) Apigenin-7-O-glycoside

331 380 381 380 385 337

288 sh 348 sh 347 340 352 sh 265

265 302 296 295 265 . . .

. . . 270 272 274 . . . . . .

(3) Luteolin-7-O-diglycoside

347 392 428 386 407 369

265 sh 263 328 357 365 sh 260

250 . . . 296 sh 295 sh 265 253 sh

. . . . . . 275 272 257 sh . . .

(4) Luteolin-4`-O-methyl (Diosmetin)

344 382 384 355 343 342

289 270 360 295 271 270

270 . . . 295 276 . . . . . .

. . . . . . 273 . . . . . . . . .

(5) Diosmetin-7-O-glycoside

343 372 382 383 340 342

265 300 sh 360 355 264 sh 264 sh

250 266 292 sh 293 sh 255 254 sh

. . . . . . 272 269 . . . . . .

(6) Diosmetin-7-O-diglycoside

343 370 381 383 340 342

267 300 sh 360 sh 360 sh 282 sh 264 sh

251 266 293 sh 293 sh 264 sh 253

. . . . . . 270 269 253 . . .

(7) Kaempferol-3-O-glycoside

352 403 397 395 372 352

295 sh 326 350 346 302 295

265 274 304 303 275 265

. . . . . . 274 274 . . . . . .

Table 3: UV spectral data of isolated flavonoid compounds (ʎmax, nm); sh=shoulder.

Austin J Plant Biol 2(1): id1014 (2016) - Page - 05

Moubasher H Austin Publishing Group

Submit your Manuscript | www.austinpublishinggroup.com

(8) Kaempferol-3-O-diglycoside

352 406 403 400 393 352

320 sh 325 358 346 312 320 sh

267 278 304 302 277 295 sh

. . . . . . 276 275 . . . 266

(9) Kaempferol-3,7-O-diglycoside

352 392 394 394 400 352

315 sh 350 sh 353 348 358 sh 300 sh

263 300 sh 300 sh 300 sh 265 268

. . . 268 270 270 . . . . . .

(10) Quercetin

372 419 452 430 373 386

306 sh 331 337 362 256 260

256 . . . 271 267 . . . . . .

(11) Quercetin-3-O-glycoside

357 405 428 400 390 377

295 326 301 sh 358 271 288 sh

266 sh 267 273 29 sh . . . 260

255 . . . . . . 269 . . . . . .

(12) Quercetin-3-O-rutinoside (Rutin)

358 417 429 400 363 380

306 sh 328 275 364 267 263

259 273 . . . 299 . . . . . .

. . . . . . . . . 270 . . . . . .

(13) Quercetin-3,7-O-diglycoside

354 430 433 400 437 380

266 sh 277 300 sh 356 385 sh 282 sh

255 . . . 273 298 262 265

. . . . . . . . . 268 . . . . . .

(14) Quercetin-3-O-diglycoside-7-O-glycoside

356 410 438 400 418 375

295 sh 269 330 sh 364 sh 375 sh 262

268 sh . . . 298 sh 298 sh 297 sh . . .

258 . . . 273 268 263 . . .

(apigenin) appeared as purple spot on 1D and 2D paper chromatogram under UV light and with ammonia fumes, whereas the aglycone: quercetin showed yellow spot with UV light, that changed to bright yellow when fumed with ammonia (Table 2). The methylation of C-4` position with and without glycosidation at C-7 appeared as purple spots on paper chromatogram upon exposure to UV light and after fumed with ammonia. Additionally, most of the other glycosides appeared as purple spots with UV light and changed to yellow with ammonia fumes.

The characteristic Rf values of each compound on 1D paper chromatograms in different solvent systems (Table 2) indicated the position of glycosides attachment. The mobility of a glycoside is closely correlated with the number and position of sugar substitution. Increasing glycosylation at C-3 position decreased Rf value in BAW and increased the mobility in 15% acetic acid relative to its aglycone. The 3,7-diglycosidation behave like the 3-glycosidation, but moved very slower in BAW and faster in 15% acetic acid. In addition, increasing glycosidation at C-7 position with methylated C-4` of luteolin showed faster movement in 15% acetic acid and lower Rf value in BAW relative to its aglycone (diosmetin).

Ultra Violet spectra (UV): The UV spectral data of isolated flavonoid compounds are shown in (Table 3). In addition, the

chemical structures of the isolated flavone and flavonol compounds are illustrated in (Table 4).

Distribution patterns of the isolated flavonoids The qualitative and quantitative analyses of the 14 flavonoids

among the five Galium species that collected from different locations of Libya are represented in (Table 5). The flavonoid patterns between the Galium species showed a predominant flavonol glycoside being quercetin-3-rutinoside (12). However, the other compounds were variable in quality, quantity, and in their distribution among the investigated samples.

In addition to the main flavonol compound (12), there are three other major flavonoids in Galium setaceum collected from Wadi Derna identified as apigenin-7-glycoside (2), kaempferol-3-glycoside (7) and quercetin-3-glycoside (11), accompanied by less major amount of apigenin (1) and quercetin (10), whereas kaempferol-3,7-diglycoside (9) present in trace amount. Galium aparine was represented by three samples collected from various locations of Libya showed resemble flavonoids pattern, but qualitatively different. These samples have a major quercetin-3-rutinoside (12), along with trace amount of quercetin-3-diglycoside-7-glycoside (14). Additionally, sample collected from Gasr Libya contained major amount of kaempferol-3-glycoside (7) with less major amount of apigenin (1)

Austin J Plant Biol 2(1): id1014 (2016) - Page - 06

Moubasher H Austin Publishing Group

Submit your Manuscript | www.austinpublishinggroup.com

and its 7-glycoside (2). However, G. aparine was collected from Shahat Ruins showed less major amounts of luteolin-7-diglycoside (3), kaempferol-3-glycoside (7) and quercetin-3-glycoside (11), along with trace amount of quercetin (10). On the other hand, apigenin (1), luteolin-7-diglycoside (3) and quercetin-3-glycoside (11) were isolated from G. aparine that collected from Wadi qaam (khoms) in less major amount, but kaempferol-3,7-diglycoside (9) is present in trace value.

With respect to Galium tricornutum, qualitative and quantitative variations of flavonoid profiles characterized by the presence of luteolin-7-diglycoside (3), diosmetin (4) and its 7-glycoside (5) and 7-diglycoside (6) with complete absence of apigenin (1) and its 7-glycoside (2), as well as the 3-glycoside of both kaempferol (7) and quercetin (11). Sample collected from Stwa afforded seven flavonoids including major amount of compounds (3), (4) and (12), along with lesser content of glycosides (8) and (9), while the flavonoids (5) and (10) detected in trace amount. There are two major flavonoids: (3) and (12) in G. tricornutum collected from Lamluda, in addition to less major compounds of (4), (5), (9) and (14) along with trace level of compounds (8) and (10). On the other hand, the major flavonoids isolated from G. tricornutum collected from Almansoura are diosmetin-7-glycoside (5) and quercetin-3-rutinoside (12), along

No. Compound R1 R2 R3 R4

1 Apigenin H OH OH H

2 Apigenin-7-O-glycoside H O-monoglycosyl OH H

3 Luteolin-7-O-diglycoside H O-diglycosyl OH OH

4 Luteolin-4`-O-Me (Diosmetin) H OH O-CH3 OH

5 Diosmetin-7-O-glycoside H O-monoglycosyl O-CH3 OH

6 Diosmetin-7-O-diglycoside H O-diglycosyl O-CH3 OH

7 Kaempferol-3-O-glycoside O-monoglycosyl OH OH H

8 Kaempferol-3-O-diglycoside O-diglycosyl OH OH H

9 Kaempferol-3,7-O-diglycoside O-monoglycosyl O-monoglycosyl OH H

10 Quercetin OH OH OH OH

11 Quercetin-3-O-glycoside O-monoglycosyl OH OH OH

12 Quercetin-3-O-rutinoside (Rutin) O-rhamnoglucosyl OH OH OH

13 Quercetin-3,7-O-diglycoside O-monoglycosyl O-monoglycosyl OH OH

14Quercetin-3-O-

diglycoside-7-O-glycoside

O-diglycosyl O-monoglycosyl OH OH

Table 4: Chemical structure of isolated flavonoids from the five investigated Galium species.

with less major amount of flavone derivatives (3) and (6), as well as flavonol glycosides (8), (13) and (14). Galium verrucosum and G. murale showed a nearly similar pattern of flavonoid compounds, particularly the methylated luteolin (diosmetin) (4), its 7-glycoside (5) and 7-diglycoside (6); with complete absence of the aglycones and simple substituted compounds including apigenin (1) and its 7-glycoside (2); luteolin-7-diglycoside (3); kaempferol-3-glycoside (7); quercetin (10) and its 3-glycoside (11). The flavonoid patterns of G. verrucosum collected from Almansoura characterized by the presence of higher amount of compounds (4), (9) and (12), accompanied by lesser content of compounds (8) and (14), as well as trace amount of flavonoid (13). In addition, sample collected from Wadi Derna contained higher levels of compound (4) and (12), with less major content of compounds (5) and (9) and trace level of flavonoid (13). On the other hand, G. verrucosum collected from Wadi qaam (khoms) has major amounts of flavonoid (5), (12) and (14), along with less major amount of compounds (6) and (13), while compound (8) present in trace level.

The two samples of Galium murale collected from Wadi Elkouf and Sharshara (Tarhuna) contained nearly similar flavonoid patterns with respect to the predominant of diosmetin-7-diglycoside (6), quercetin-3-rutinoside (12) and quercetin-3-diglycoside-7-glycoside (14), along with less major amount of compound (9). However, the quercetin-3,7-diglycoside (13) was isolated only in the first sample that replaced by diosmetin-7-glycoside (5) in the second sample only.

Multivariate analyses: Morphologically similar and taxonomically closer species have similar even same flavonoid profiles. So, they clustered together in the dendrogram. Three groups of species can be separated in the dendrogram (Figure 1). The PCA biplot in (Figure 2) showed that each group is correlated to more than one compound.

Chemotaxonomic significance: This study confirmed that flavonoid content is a useful marker in the taxonomy of Galium. Kaempferol-3,7-O-diglycoside and Quercetin-3-O-rutinoside (Rutin) are found in all species. The isolated and structure elucidation of flavonoid compounds afford the presence of simple and relatively complex flavonoids identified among the five Galium species. The flavonoid complexity appeared in the substitution patterns of flavone and flavonol aglycones with O-glycosylated and O-methylated attachments. Generally, the flavonoid chemistry in all studied Galium species appears to be relatively homogeneous based on the predominance of flavonol glycoside, particularly quercetin-3-rutinoside (12).

The first group (group C) included Galium setaceum and G. aparine, which are characterized by simple O-glycoside of apigenin, kaempferol and quercetin. However, the detection of luteolin-7-diglycoside (3) along with trace amount of complex O-glycosylated quercetin (14) only in Galium aparine and their lacking from G. setaceum indicates the more specialized of the former species relative to the second one. Galium verrucosum and G. murale represent the second group (group B), which was encountered to produce methylated flavone: diosmetin (4) and its glycosidic attachments (5) and (6), along with the presence of complex O-glycosylated kaempferol and quercetin, particularly a C-3 and C-7 positions (9 and 14). In addition, the frequent lacking of simple aglycones of apigenin

Austin J Plant Biol 2(1): id1014 (2016) - Page - 07

Moubasher H Austin Publishing Group

Submit your Manuscript | www.austinpublishinggroup.com

(1) and quercetin (10), as well as their simple glycosidic forms (2), (3), (7) and (11) indicates that both species are of evolutionary advanced than the two species of first group.

With respect to their classification, Group (A) comprised of Galium verrucosum (S9, S10) and G. tricornutum (S2, S7). Morphologically, these two species are linked together as they share some characters such as mericarp size, petal length, flower diameter size, and petal width. They are clustered together as a result of the same flavonoid profiles (Figure 2). Group (B) included G. murale (S5, S8), G. verrucosum (S11) and G. tricornutum (S3) as well. The former species is characterized by its seed and mericarp shapes. Group (C) included two different species: G. setaceum (S6) and G. aparine (S1, S4, S12). Both species are correlated with petal colour, style length, leaf width, leaf shape, and pedicel length. Remarkably, G. aparine (S1, S4, S12) that collected from distant mountainous and isolated areas do not affected by any flavonoid except for the two which are common to all species. The phytochemical results are relatively congruent with morphological data.

However, qualitative and quantitative variations in the flavonoid profiles among the five investigated Galium species collected from Libya could respond to the environment-dependent variability [24]. Such change may be related to genetic variation amongst individuals that affected by environmental factors. On the other hand, Puff [25] described that variation change in the quality and quantity in the flavonoid profiles in the most taxa of Galium sect. aparinoides is related to change of the environment. Separate publications on the effect of some environmental variables, and genetic variations among the studied Galium are in preparation.

In Conclusion, the flavonoid patterns in the Galium species showed a predominant flavonol glycoside being quercetin-3-rutinoside [26]. However, the other compounds were variable in quality, quantity and in their distribution among the investigated samples. The distribution patterns of the isolated fourteen flavonoid compounds showed the presence of biosynthetic divergence in the

CompoundG. setaceum G. aparine G. tricornutum G. verrucosum G. murale

S6 S4 S1 S12 S7 S2 S3 S9 S10 S11 S5 S8

1 - Apigenin + + - + - - - - - - - -

2 - Apigenin-7-O-glycoside ++ + - - - - - - - - - -

3 - Luteolin-7-O-diglycoside - - + + ++ ++ + - - - - -

4 - Luteolin-4`-O-Me (Diosmetin) - - - - ++ + - ++ ++ - - -

5 - Diosmetin-7-O-glycoside - - - - t + ++ - + ++ - +

6 - Diosmetin-7-O-diglycoside - - - - - - + - - + ++ ++

7 - Kaempferol-3-O-glycoside ++ ++ + - - - - - - - - -

8 - Kaempferol-3-O-diglycoside - - - - + t + + - t - -

9 - Kaempferol-3,7-O-diglycoside T - - t + + - ++ + - + +

10 - Quercetin (Q) + - t - t t - - - - - -

11 - Q-3-O-glycoside ++ - + + - - - - - - - -

12 - Q-3-O-rutinoside (Rutin) ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++

13 - Q-3,7-O-diglycoside - - - - - - + T t + + -

14 - Q-3-O-diglycoside-7-O--glycoside - t t t - + + + - ++ ++ ++

Table 5: The distribution of flavonoids in Galium species collected from different localities of libya. For localities of Galium species.

glycosidic and methylated attachments that possible to distinguish between two main groups with an intermediate group.

References1. Mabberley DJ. The Plant-Book, Cambridge University Press, Cambridge,

New York, Melbourne. 1997.

2. Dempster LT, Delprete PG, Smith LB, Klein RB. Rubiáceas. Galium. Flora ilustrada catarinense. Herbário Barbosa Rodrigues, Itajaí. 2004; 273-342.

3. Schischkin BK. Flora of the USSR, Delhra Dunn, India: Bishen Singh Mahendra Pal Singh. Koeltz Scientific Books, Koenigstein. 2000.

4. Borisov MI, Komissarenko NF. Flavonoids of Galium palustre. Chemistry of Natural Compounds. 1969; 5: 371-375.

5. Chen LJ, Bin LL, Shi Jun L, Jun Xing D. Study on the chemical constituents of Galium aparine L. Journal of International Pharmaceutical Research. 2010; 37: 387-389.

6. El-Gamal AA, Koichi T, Itokawa H, Halim AF, Amer MM, Saad HEA, et al. Anthraquinones from the polar fractions of Galium sinaicum. Phytochemistry. 1995; 40: 245-251.

7. Schildknecht H, Straub F, Scheidel V. Mollugin a new pigment from rhizomes of Galium mollugo. Justus Liebigs Annals of Chemistry. 1976; 8: 1295–1306.

8. Borisov MI. Flavonoids of Galium ruthenicum. Chemistry of Natural Compounds. 1974; 10: 662-663.

9. Iavarone C, Sen A, Trogolo C, Villa S. Mollugoside, an iridoid glucoside from Galium mollugo. Phytochemistry. 1983; 22: 175-178.

10. Zhao CC, Shao JH, Li X, Kang XD, Zhang YW, Meng DL, et al. Flavonoids from Galium verum L. Journal of Asian Natural Products Research. 2008. 10: 611-615.

11. Zhao CC, Shao JH, Cao DD, Zhang YW, Li X. Chemical constituents of Galium verum L. China Journal of Chinese Materia Medica. 2009; 34: 2761-2764.

12. Corrigan D, Timoney R, Donnelly D. Iridoids and alkanes in twelve species of Galium and Asperula. Phytochemistry. 1978; 17: 1131-1133.

13. Uesato S, Miyauchi M, Itoh H, Inouye H. Biosynthesis of iridoid glucosides in Galium mollugo, Galium spurium var. echinospermon and Deutzia crenata. Intermediacy of deoxyloganic acid, loganin and iridodial glucoside. Phytochemistry. 1986; 25: 2515–2521.

Austin J Plant Biol 2(1): id1014 (2016) - Page - 08

Moubasher H Austin Publishing Group

Submit your Manuscript | www.austinpublishinggroup.com

14. Baytop T. Therapy with Medicinal Plants in Turkey: Past and Present. Istanbul University Publications. 1984.

15. Grzybek J, Wongpanich V, Mata-Greenwood E, Angerhofer CK, Pezzuto JM, Cordell GA. Biological evaluation of selected plants from Poland. International Journal of Pharmacognosy. 1997; 35: 1-5.

16. Boulos L, Barakat HN, Hegazy AK. Endemic Flora of the Middle East and North Africa. Reviews in Ecology: Desert Conservation and Development. 1997; 229-260.

17. Brullo S, Furnari F. La vegetation del Gebel el-Akhdar (Cirenaica settentrionale). Boll Acc Gioenia Sci Nat. 1994; 27: 197-412.

18. Le Houérou HN. Biogeography of the arid steppeland north of the Sahara. Reviews in Ecology: Desert Conservation and Development. Cairo: Metropole, Egypt. 1997; 207-228.

19. Feng Y, Lei J, Xu X, Pan R. Composition and characteristics of Libyan flora. Archives of Biological Sciences, Belgrade. 2013; 65: 651-657.

20. Soltis D, Soltis PS, Endress PK, Chase MW. Phylogeny and Evolution of Angiosperms. Sinauer, Sunderland. 2005.

21. Mabry TJ, Markham KR, Thomas MB, Mabry TJ, Markham KR, Thomas MB. The Systematic Identification of Flavonoids. Springer-Verlag and New York. 1970.

22. Hammer Ø, Harper DAT, Ryan PD. PAST: Palentological Statistics software package for education and data analysis. Palaentologia Electronica. 2001; 4: 9.

23. Kovach WL. A Multivariate Statistical Package for Windows, Manual. Kovach Computing Services, Pentraeth, Wales, UK. 1999.

24. Ehrendorfer F, Schönbeck-Temesy E, Puff C, Rechinger W. Rubiaceae Rechinger, Flora Iranica. Verlag des Naturhistorischen Museums Wien, Vienna, Austria. 2005.

25. Harborne JB. Comparative Biochemistry of the Flavonoids. Academic Press, London and New York. 1967.

26. Puff C. Leaf flavonoids of Galium Sect. Aparinoides (Rubiaceae). Plant Systematics and Evolution. 2001; 124: 57-66.

Citation: Moubasher H, Abd El-Ghani M, Al-Wakeel S and Bahoor A. Chemotaxonomic Significance of Flavonoids in Some Species of Galium (Rubiaceae) from Libya. Austin J Plant Biol. 2016; 2(1): 1014.

Austin J Plant Biol - Volume 2 Issue 1 - 2016ISSN: 2472-3738 | www.austinpublishinggroup.com Moubasher et al. © All rights are reserved